The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of checkpoint controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2, M) which correlate with distinct cell morphology and biochemical activity, and cells withdrawn from the cell-cycle are said to be in G0, or non-cycling state. When cells within the cell-cycle are actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of organization that result in the formation of chromosomes. Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 5, 1997).
Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989, Science, 246: 629-634). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997, Science, 274: 1664-1671). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et al., 1997, Mol. Cell Biol., 8: 1-11; Jin et al., 1996, J. Cell Biol., 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997, Cancer Res., 57: 5168-5178).
Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the G1-S transition (Hoffmann et al., 1994, EMBO J., 13: 4302-4310; Jinno et al., 1994, EMBO J., 13: 1549-1556), whereas Cdc25B and Cdc25C are required for the G2-M transition (Gabrielli et al., 1996, J. Cell Sci., 7: 1081-1093; Galaktionov et al., 1991, Cell, 67: 1181-1194; Millar et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol., 138: 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known.
Much of our current knowledge about checkpoint control has been obtained from studies using budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. A number of reviews of our current understanding of cell cycle checkpoints in yeast and higher eukaryotes have recently been published (Hartwell & Kastan, 1994, Science, 266: 1821-1828; Murray, 1994, Current Biology, 6: 872-876; Elledge, 1996, Science, 274: 1664-1672; Kaufmann & Paules, 1996, FASEB J., 10: 238-247). In the fission yeast six gene products, rad1+, rad3+, rad9+, rad 17+, rad26+, and hus1+ have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cds1+ has been identified as being required for the DNA-replication dependent checkpoint and rad27+/chk1+ has been identified as required for the DNA-damage dependent checkpoint in yeast.
Several of these genes have structural homologues in the budding yeast and further conservation across eukaryotes has recently been suggested with the cloning of two human homologues of S. pombe rad3+: ATM (ataxia telangiectasia mutated) (Savitsky et al., 1995, Science, 268: 1749-1753) and ATR (ataxia telangiectasia and rad3+ related)(Bentley et al, 1996, EMBO J., 15: 6641-6651; Cimprich et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 2850-2855) and of a human homologue of S. pombe rad9+ (Lieberman et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 13890-13885).
While much is known about yeast checkpoint proteins and genes, this knowledge is not fully predictive of the existence of corresponding human genes or proteins, or their effector role in human cell-cycle control and regulation.
In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of cancer, it is important to identify and characterize human checkpoint proteins and to identify mediators of their activity.